In vitro bone formation by rat marrow cell culture

Ohgushi, Hajime; Tamai, Susumu; Dohi, Yoshiko; Katuda, Toshiya; Tabata, Shiro; Suwa, Yoshiko

doi:10.1002/(sici)1097-4636(199611)32:3<333::aid-jbm5>3.0.co;2-t

Cited by 232 publications

(178 citation statements)

References 22 publications

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“…Various in vitro bone regeneration strategies have relied on the use of the osteogenic supplements (dexamethasone, ascorbic acid, and b-glycerophosphate) to induce mineralization in vitro, 12,13,49,51,[53][54][55][56][57][58][59][60][61][62] and each supplement has been shown to be imperative to induce mineralization of MSCs in vitro. 9,[11][12][13][14][15][16][17]22,29,[63][64][65][66][67] The main rationale behind the use of these supplements is to provide signaling proteins or enzymes to activate mineral production by MSCs.…”

Section: Discussionmentioning

confidence: 99%

Osteogenic Differentiation of Mesenchymal Stem Cells by Mimicking the Cellular Niche of the Endochondral Template

Freeman

Stevens

Owens

et al. 2016

Tissue Engineering Part A

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In vitro bone regeneration strategies that prime mesenchymal stem cells (MSCs) with chondrogenic factors, to mimic aspects of the endochondral ossification process, have been shown to promote mineralization and vascularization by MSCs both in vitro and when implanted in vivo. However, these approaches required the use of osteogenic supplements, namely dexamethasone, ascorbic acid, and b-glycerophosphate, none of which are endogenous mediators of bone formation in vivo. Rather MSCs, endothelial progenitor cells, and chondrocytes all reside in proximity within the cartilage template and might paracrineally regulate osteogenic differentiation. Thus, this study tests the hypothesis that an in vitro bone regeneration approach that mimics the cellular niche existing during endochondral ossification, through coculture of MSCs, endothelial cells, and chondrocytes, will obviate the need for extraneous osteogenic supplements and provide an alternative strategy to elicit osteogenic differentiation of MSCs and mineral production. The specific objectives of this study were to (1) mimic the cellular niche existing during endochondral ossification and (2) investigate whether osteogenic differentiation could be induced without the use of any external growth factors. To test the hypothesis, we evaluated the mineralization and vessel formation potential of (a) a novel methodology involving both chondrogenic priming and the coculture of human umbilical vein endothelial cells (HUVECs) and MSCs compared with (b) chondrogenic priming of MSCs alone, (c) addition of HUVECs to chondrogenically primed MSC aggregates, (d-f) the same experimental groups cultured in the presence of osteogenic supplements and (g) a noncoculture group cultured in the presence of osteogenic growth factors alone. Biochemical (DNA, alkaline phosphatase [ALP], calcium, CD31 + , vascular endothelial growth factor [VEGF]), histological (alcian blue, alizarin red), and immunohistological (CD31 + ) analyses were conducted to investigate osteogenic differentiation and vascularization at various time points (1, 2, and 3 weeks). The coculture methodology enhanced both osteogenesis and vasculogenesis compared with osteogenic differentiation alone, whereas osteogenic supplements inhibited the osteogenesis and vascularization (ALP, calcium, and VEGF) induced through coculture alone. Taken together, these results suggest that chondrogenic and vascular priming can obviate the need for osteogenic supplements to induce osteogenesis of human MSCs in vitro, while allowing for the formation of rudimentary vessels.

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Section: Discussionmentioning

confidence: 99%

Osteogenic Differentiation of Mesenchymal Stem Cells by Mimicking the Cellular Niche of the Endochondral Template

Freeman

Stevens

Owens

et al. 2016

Tissue Engineering Part A

View full text Add to dashboard Cite

show abstract

“…1E0666, Lenexa, KS, USA). Rat MSCs were isolated and maintained in primary culture as previously described (19,20). Briefly, bone marrow cells were obtained from the femoral bone shaft of 7-week-old male F344 rats and seeded into 75 cm 2 flasks (T-75 flasks, Corning Costar, Cambridge, MA, USA) containing 15 ml of standard medium consisting of MEM supplemented with 15% FBS and a mixture of antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin; Sigma-Aldrich, St. Louis, MO, USA).…”

Section: Methodsmentioning

confidence: 99%

Effect of mesenchymal stem cells on hypoxia-induced desensitization of β2-adrenergic receptors in rat osteosarcoma cells

et al. 2012

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Abstract. The β2-adrenergic receptor (β2AR) mediates the effects of chronic stress in several neoplasms, however, β2AR signaling is impaired by hypoxia in various tissues. While hypoxia is a common feature significant in the progression of solid tumors, little is known about the effect of hypoxia on β2AR signaling in the tumor microenvironment. Previously, it has been reported that the systemic administration of mesenchymal stem cells (MSCs) increased the engraftment and metastatic colonization of rat osteosarcoma (OS) cells. In the current study, the effect of MSCs on the hypoxia-induced desensitization of the β2AR in OS cells was investigated. Epinephrine, norepinephrine and isoproterenol increased the cellular proliferation of the rat OS cell line COS1NR and rat MSCs in a dose-dependent and β2AR antagonist-sensitive manner. While isoproterenol had significant proliferative effects on MSCs under normoxic and hypoxic conditions, COS1NR cells did not respond under hypoxic conditions. A sensitivity assay for the β2AR revealed that hypoxia impaired the sensitivity of COS1NR cells, whereas hypoxia did not affect MSCs. An immunoassay revealed no significant change in the expression of hypoxia-inducible factor-1α (HIF1α) in COS1NR cells, whilst an immunoassay demonstrated a 15% increase in MSCs following isoproterenol stimulation. In COS1NR cells co-cultured with MSCs under hypoxic conditions, isoproterenol caused a significant increase in proliferation and this effect was inhibited by an anti-interleukin (IL)-6 antibody. A tumor formation assay in syngeneic rats revealed that the systemic administration of MSCs enhances the growth of OS and the effect of MSCs was inhibited by IL-6 neutralization.In conclusion, MSCs are resistant to the hypoxia-induced desensitization to β2AR. Hypoxia caused a siginificant desensitization of the β2AR in COS1NR cells alone, whereas MSCs may support tumor progression through cellular interactions.

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“…15,16 Briefly, fresh bone marrow plugs were obtained from the femoral shafts of 7-week-old male Fischer 344 rats and flushed out using 10 mL of culture medium expelled gently from a syringe through a 21-gauge needle. The released cells were collected in a T-75 flask (Coster Co., Cambridge, MA) containing 15 mL of the standard medium described above.…”

Section: Marrow Mesenchymal Stem Cell Preparation and Culture Conditionmentioning

confidence: 99%

Recombinant growth/differentiation factor‐5 (GDF‐5) stimulates osteogenic differentiation of marrow mesenchymal stem cells in porous hydroxyapatite ceramic

Shimaoka

Dohi

Ohgushi

et al. 2003

J Biomedical Materials Res

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Abstract:To evaluate the growth/differentiation factor-5 (GDF-5) in the in vivo osteogenic potential of bone marrow mesenchymal stem cells (MSCs), we subcutaneously implanted five different kinds of hydroxyapatite (HA) ceramic implants: HA alone, GDF-5/HA composites (GDF/HA), MSCs/HA composites, the MSCs/HA composites supplemented with GDF-5 (GDF/MSCs/HA), and recombinant bone morphogenetic protein-2 (BMP/MSCs/HA). Neither the HA alone nor the GDF/HA composites exhibited any bone formation at any time after implantation. At 4 weeks, the MSCs/HA composites exhibited a certain amount of bone formation in some pore areas. In contrast, at 2 weeks, the GDF/MSCs/HA composites exhibited histologically obvious de novo bone formation together with active osteoblasts in many pore areas and additional bone formation at 4 weeks. In the de novo formed bone, neither chondrocytes nor endochondral bone was detected. The GDF/MSCs/HA composites also showed high alkaline phosphatase (ALP) and osteocalcin expression determined at both the protein and gene levels and the high level of expression was well maintained even at 4 weeks. Compared with GDF/MSCs/ HA, the BMP/MSCs/HA composites exhibited excellent osteogenesis with relatively early osteoblastic phenotype expression. The results indicate that GDF-5 synergistically enhances de novo bone formation capability of MSCs/HA composite and suggest that tissue-engineered GDF/ MSCs/HA composites could be used as bone graft substitutes.

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In vitro bone formation by rat marrow cell culture

Cited by 232 publications

References 22 publications

Osteogenic Differentiation of Mesenchymal Stem Cells by Mimicking the Cellular Niche of the Endochondral Template

Osteogenic Differentiation of Mesenchymal Stem Cells by Mimicking the Cellular Niche of the Endochondral Template

Effect of mesenchymal stem cells on hypoxia-induced desensitization of β2-adrenergic receptors in rat osteosarcoma cells

Recombinant growth/differentiation factor‐5 (GDF‐5) stimulates osteogenic differentiation of marrow mesenchymal stem cells in porous hydroxyapatite ceramic

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